This invention relates to biologically active wound dressings and more particularly to biologically active bilayered constructs useful as a wound dressing, as a skin equivalents or as skin substitutes. It relates also to the manufacture of such constructs in an economical, large scale, process.
Burn wounds, in general, are exceedingly painful and difficult to heal. Burns can be partial thickness burns, which destroy some, but not all of the epidermis and may destroy a portion of the dermis. Some partial thickness burn wounds will heal if treated properly with bioactive dressings, which can protect the wound and promote rapid epithelialization with minimal inflammation and scar formation. Full thickness burns, on the other hand, destroy all of the epidermis, the hair follicles, sweat glands and sebaceous glands and frequently much of the dermis. Full thickness burns ultimately require skin grafting.
Several types of skin grafts have been used to cover and repair damaged skin. Autografts are the most effective skin grafts and are tissue transplants derived from the injured individual, usually in the form of split-thickness skin grafts. A split-thickness skin graft consists of skin removed from a donor site and placed on a full thickness wound, after debridement of the dead tissue, to close and heal the wound. Split-thickness skin grafts, comprise the epidermis, part of the epidermal adnexal structures and part of the dermis. Typically, a split-thickness skin graft is meshed (short, alternating incisions) which allows for a maximum of 1:10 expansion of the graft tissue and usually an expansion of 1:3 or less. Other types of skin grafts include allografts, which are tissue transplants between individuals of the same species but different genotypes, and homografts, which are allografts from humans. Harvesting these grafts creates additional skin wounds which, in turn, need to be treated and may compromise the patient further.
Disadvantages of skin grafts, other than autografts, include infection and frequent rejection by the recipient requiring the use of immunosuppressive agents. Research efforts have been directed towards developing functional substitutes, that overcome the disadvantages of skin substitutes derived from animal skin, to provide permanent wound closure.
An effective bioactive wound dressing should facilitate the repair of wounds that may require restoration of both the epidermis and dermis. To be successful such a skin graft must be placed onto, and be accepted by, the debrided wound of the recipient and provide a means for the permanent re-establishment of the dermal and epidermal components of skin. The graft should not evoke an immune response, which can destroy the graft, and should include suitable dermal components to support the growth and development of a normal epidermis. The graft should suppress the formation of granulation tissue which causes scarring.
Additional criteria for biologically active wound dressings include: rapid adherence to the wound soon after placement; proper vapor transmission to control evaporative fluid loss from the wound and to avoid the collection of exudate between the wound and the dressing material. Skin substitutes should act as barrier to microorganisms, limit the growth of microorganisms already present in the wound, be flexible, durable and resistant to tearing. The substitute should exhibit tissue compatibility, that is, it should not provoke inflammation or foreign body reaction in the wound which may lead to the formation of granulation tissue. An inner surface structure should be provided that permits ingrowth of fibro-vascular tissue. An outer surface structure should be provided to minimize fluid transmission and promote. epithelialization. A variety of materials and constructions have been proposed to meet these requirements.
Synthetic polymeric materials in various forms have been tested for the development of skin structures having the ability to induce cellular migration and proliferation into the graft. This effort has been limited by the high incidence of infection and inability to promote vascularization and epithelialization. Epithelialization of the membrane graft provides a barrier to infection and contributes to the control of fluid loss.
Typical bioabsorbable materials for use in the fabrication of porous wound dressings, skin substitutes and the like, include synthetic bioabsorbable polymers such as polylactic acid or polyglycolic acid, and also, biopolymers such as the structural proteins and polysaccharides. Skin substitutes made from synthetic polymers have, for a number of reasons, met with limited success. The structural proteins have also met with limited success and include collagen, elastin, fibronectin, laminin and fibrin, as well as other proteins of the human connective tissue matrix. Of these, the material most studied has been collagen. Collagen is the most abundant animal protein and the major protein of skin and connective tissue. A high degree of homology exists between the various types of collagen found in different animal species and human collagen. Accordingly, animal collagen types such as bovine collagen are useful because they exhibit very low immunogenicity when implanted into humans or used as topical dressings on human wounds.
However, the use of collagen alone as a reconstituted collagen film, sponge or sheet for example, has not been demonstrated to serve as an effective wound covering for various reasons among which are the stimulation of the development of granulation tissue and production of a chronic inflammatory response before being resorbed or biodegraded.
Besides films or sheets, collagen may be prepared in a variety of physical forms including porous mats and sponges. Freeze drying an aqueous gel or an aqueous suspension of collagen may be used to produce a porous collagen sponge. Such collagen sponges are described, for example, by Chvapil and co-workers in J. Biomed. Mater. Res. 11 721-741 (1977).
Porous implants, made from biological, bioabsorbable components, are normally intended to be invaded by the cells of the host or recipient of the implant. By and large, these sponges have not proven to be very useful. Later developments, using sponges of appropriate structure and inoculated with suitable cell types have, however, shown considerable promise.
The prior art processes for preparation of a cell-impermeable film on a surface of lyophilized collagen sponge to support and anchor a cellular component, e.g. keratinocytes, generally done using complex and technically difficult procedures. Earlier work by others in this field includes the following:
Yannas, (U.S. Pat. No. 4,060,081) teaches the preparation of a fibrous layer of a mixture of collagen and chondroitin-6-sulfate (GAG) to which is attached a silicone component. The collagen/GAG component of this skin substitute was found to be biodegradable and was said not to be inflammatory or immunogenic. However, it required that the silicone xe2x80x9cepidermisxe2x80x9d be removed at a later date and that the dermal layer be covered with a thin autograft, to provide the epidermal component, for permanent wound closure.
In U.S. Pat. No. 4,505,266, Yannas discloses the preparation of a cross-linked, bi-layer sponge which has a silicone membrane coated on its surface to serve as a moisture barrier. A milled collagen dispersion is blended with chrondroitin 6-sulfate and the mixture poured into freezing trays. This was then lyophilized for a period of 24 to 48 hours to form a porous structure. When the lyophilization was complete, the sponge was cross-linked by heating for about 24 hours at 105xc2x0 C. Finally a silicone adhesive was coated over the entire exposed surface of the cooled foam. After curing, the silicone formed an impermeable layer.
Berg discloses a surface coating of a collagen construct with a noncollagenous, non-bioabsorbable adhesive (U.S. Pat. No. 4,841,962).
Ksander, in U.S. Pat. No. 4,950,483, discloses that multilayer atelopeptide collagen sponge products can be formed by serially casting and flash freezing each layer as it is applied. This was followed by lyophilizing and drying the plurality of layers. This process lyophilizes the combined layers and makes them porous. There does not appear to be an impermeable layer.
Silver describes a biodegradable collagen construct allegedly suitable for use as a wound implant (U.S. Pat. No. 4,970,298). The construct was formed by freeze drying an aqueous dispersion containing collagen, cross-linking the collagen via two cross-linking steps and freeze-drying the cross-linked matrix.
The preparation of a laminated, thermally cross-linked sponge consisting of a mixture of collagen and a mucopolysaccharide (GAG) is described by Boyce (U.S. Pat. No. 5,273,900). A mixed collagen and chondroitin-6-sulfate solution was deep frozen and lyophilized. It then was cross-linked at 105xc2x0 C. A mixed collagen and mucopolysaccharide solution, additionally containing 3% dimethyl sulfoxide (DMSO), was sprayed onto a flat Mylar surface, frozen and the cross-linked sponge then placed onto the frozen solution.
Yoshizato (U.S. Pat. No. 5,350,583, U.S. Pat. No. 5,263,983) discloses coating denatured atelocollagen sponges, with or without a supporting layer, with a silicone permeation controlling layer.
Rosenthal (U.S. Pat. No. 5,565,210) describes composites comprising a collagen sponge construct having embedded therein oriented substructures of solid collagen fibers, films or flakes. The substructures are oriented so as to provide a scaffold for directional cellular migration into the implant. The composites are formed by immersing the substructures in an aqueous collagen suspension and then freeze-drying the suspension to form the porous collagen sponge matrix.
In Japanese Publication No. 02-071749 (JP 2071749A, application no. JP 88222538), inventor Koide Mikio, published Mar. 12, 1990, a porous collagen layer on top of a porous collagen/denatured collagen substrate is described. Both layers are porous to allow the transmission of nutrients and fluids from a wound surface to the top of the dressing.
Eisenberg, in U.S. Pat. No. 6,039,760, U.S. Pat. No. 5,282,859, and RE 35,399, discloses a process for preparing a collagen sponge that has been cross-linked by thermal dehydration. The sponge is laminated on one surface with a thin layer of high purity, preferably pepsin treated, non-porous collagen. The sponge and the layer are then dried to complete the lamination of the collagen. The sponge is then inverted and inoculated with fibroblast cells and allowed to culture. The sponge is inverted once again and keratinocytes are inoculated on the non-porous layer and allowed to culture.
The present invention relates to a collagen construct having a structure suitable for use as a biologically active skin wound dressing or a skin equivalent, for example, and the process for preparing said construct. Prior to actual use as a biologically active wound dressing or skin equivalent, the construct is inoculated with appropriate cell types in and/or on the surface thereof that produce growth factors and other bioactive substances. Such biologically active products can be used in many different applications that require the regeneration of dermal tissues. They have been used in the repair of injured skin and difficult-to-heal wounds, such as burn wounds. venous stasis ulcers or diabetic ulcers. The essence of the construct of the present invention is the presence of a cell-impermeable transitional collagen layer which, as will be seen later, acts as a barrier layer, between and joining two layers of collagen, one of which is a porous sponge layer and the other a thin surface forming the outer boundary of the transitional layer. Most preferably, the cell-impermeable transitional layer has deposited thereon a very thin layer of acid-soluble collagen which acts as a cell attachment layer. Thus, the construct in its basic form, is a bilayered device which in its most preferred form carries a thin layer as a site for cell attachment. The process of the present invention is a cost effective, efficient process amenable to large-scale manufacture of the skin substitute.
The term xe2x80x9cbilayeredxe2x80x9d as used herein is intended to mean a construct comprising the collagen porous sponge attached to a cell-impermeable transitional or barrier collagen layer having a boundary surface. The boundary surface is the outer surface of the transitional layer opposite to the interface between the sponge and the transition layer. The bilayered construct may or may not have a cell attachment layer on the boundary layer.
The term xe2x80x9cnon-porousxe2x80x9d as used herein is intended to mean that the object so referred to is impermeable to the migration of human cells therethrough.
Referring to the construct of this patent, the terms xe2x80x9cpermeablexe2x80x9d and xe2x80x9cimpermeablexe2x80x9d as applied to the sponge and its transition layer, refer to materials which have pores large enough to be ingrown by cells (permeable) and, respectively, pores, if any, which are small enough so that cells can not migrate through them (impermeable).
The term xe2x80x9cspongexe2x80x9d is intended to mean a structure having spaces therein capable of permitting the migration and growth of human fibroblasts therein.
To manufacture large numbers of such devices economically, the present invention permits the use of a larger sponge than is normally used in the art, i.e., about one by two feet, which can be divided into smaller pieces of skin equivalent after the cell culturing process is completed.
The product of the present invention is a lyophilized construct comprising a collagen sponge, preferably bovine collagen, and a cell-impermeable layer having a boundary surface with the boundary surface being separated from the sponge section by the transitional layer which is produced when the sponge is placed on a collagen dispersion cast on a flat plate and dried. Most preferably, the boundary surface has deposited thereon a thin cell attachment layer. The thin cell attachment layer is comprised of acid-soluble collagen and acts to provide a keratinocyte-hospitable xe2x80x9cthirdxe2x80x9d layer of the construct. A commercially available, sterile bovine collagen (VITROGEN(trademark)) suitable for the third layer is available from Cohesion, Inc. of Calif. The bilayered construct is preferably DHT cross-linked after formation. If the sponge layer portion has been previously cross-linked, the transitional and the thin acid soluble collagen cell attachment layer may be subsequently cross-linked using other means such as ultraviolet radiation. The finished bilayered sponge (with or without the cell attachment layer) is packaged and preferably radiation sterilized. Appropriate cells such as human keratinocytes can then be cultured on the cell attachment layer and human fibroblasts cultured in the sponge matrix as described herein.
The present invention thus involves new bilayered collagen constructs and new processes therefore wherein the surface of a preformed sponge is partially xe2x80x9cmeltedxe2x80x9d or compacted into a collagen dispersion, under controlled conditions, to yield a construct in which the sponge becomes fused to and intermingled with a cell-impermeable transition layer of desired or controlled depth connected to the sponge, the transition layer terminating in a boundary surface which may or may not be, but most preferably is, further enhanced with a cell attachment layer deposited thereon.
The reason that a cell attachment layer is sometimes used, is that the boundary surface of the transitional layer may not be suitable for some purposes involving keratinocyte cell attachment. In fact, in the present invention, it is most preferred that a separate cell attachment layer comprising a very thin coating of keratinocyte-compatible, enzymatically solubilized acid-soluble collagen such as VITROGEN(trademark) be present on the boundary surface of the transitional layer and stabilized by drying and/or by cross-linking, thus providing a xe2x80x9cthirdxe2x80x9d layer. Large amounts of expensive VITROGEN(trademark), otherwise needed to form a cell-impermeable barrier, are avoided by virtue of the present invention which provides a cell-impermeable transition layer that may or may not (depending upon the cell type) require thin acid soluble collagen layer, thus effecting substantial cost savings.
The new process is simpler than the art process and does not require complex, labor intensive and time consuming processing under aseptic conditions preventing economical production of large amounts of bilayer sponge. The new process avoids problems such as gel invagination into the porous sponge and incomplete barrier formation that may occur using the art-known processes.